Deletion mutant of a herpesvirus and vaccine containing said virus

ABSTRACT

The invention relates to live deletion mutants of a herpesvirus, especially of Pseudorabies virus. The genomes of the mutants differ from the genomes of the parent strain by the presence of one or more deletions selected from a deletion in the inverted repeats, a deletion in the unique sequence, and a deletion in the thymidine kinase gene. 
     The new deletion mutants are avirulent, do not induce latent infections, are stable and possess good immunogenic properties. 
     The invention also relates to vaccines containing the new mutant strains.

The invention relates to a live deletion mutant of a herpesvirus,especially to a virus derived from pseudorabies virus (PRV), also calledAujeszky's disease virus (ADV). Psuedorabies is a disease of alldomestic animals, with the exception of the horse, and causes severedamage, especially among pigs and cattle. The pig is the natural host ofthe herpesvirus of Aujeszky's disease. The animals are infected via thenasal route and, after a primary virus multiplication in the mucousmembranes of upper respiratory and digestive tracts, the virus spreadsvia nerves to the brain. The infection proceeds acutely tosub-clinically, which is mainly dependent on the virulence of the virusand the age of the pigs. Pseudorabies virus, just as other herpesvirusesinduces latent infections, namely in the nerve tissues.

Vaccinations are carried out so as to limit somewhat the economicaldamage caused by mortality and growth retardation. For this purposevaccines are available which are based on attenuated live virus and oninactivated virus, respectively. The vaccines based on attenuated livevirus are more .[.simply.]. .Iadd.simple .Iaddend.to produce andtherefore cheaper than the vaccines based on inactivated virus.Moreover, the vaccines containing attenuated live virus have theadvantage that they can also be used via the intranasal route. Theintranasal inoculation results in a better protection against thedisease than a parenteral vaccination with attenuated live virus or withinactivated virus.

The attenuated live virus vaccines presently in use possess variousdisadvantages:

(1) They are generally produced by serial passages of virulent or mildlyvirulent strains in tissue culture (50-900 passages). This inducesuncontrolled mutations in the viral genome.

(2) The composition of most of the commercial vaccines is nothomogeneous. Namely, the vaccines contain a number of virus variants,the virulence of which and the vaccinating power of which are notdefined.

(3) There is a danger of return to virulence.

(4) There is a risk of the induction of latent infections.

The genome structure of the DNA of pseudorabies virus (PRV) has beendescribed in the literature (Virology 97 (1979), pages 151-163). On thebasis of the arrangement of the reiterated sequences PRV belongs to thegroup of D-herpesviruses. They contain a genome of about 160,000nucleotide pairs containing two inverted repeats and two uniquesequences, called U_(s) and U₁. The virus occurs in two isomeric forms,both of which are infectious. In order to obtain insight in thestructure of the genome and in the variability between the many isolatesavailable, the DNA's of a number of vaccine strains and of some 100independent PRV isolates obtained in Holland and other countries fromanimals suffering from the disease were compared by means of restrictionmapping. This analysis, which was carried out with a number ofrestriction enzymes, showed the following:

(a) The individual isolates show a high degree of sequential homology,that is to say a nick-translated probe of the virus NIA-3 (NorthernIreland Aujeszky-3) used as a reference recognized, under stringenthybridization conditions, all fragments of the field isolates.

(b) The structures of the various isolates show a high mutualsimilarity. Generally, corresponding sequences are located in the sameareas.

(c) Certain regions show more variability than other. For example, theoverlap fragments of inverted repeat to unique sequence showed a strongheterogeneity.

(d) The strains used in the vaccines, although mostly heterogeneous withrespect to the DNA composition and poorly definable, contain a deletionvarying from 1000 to 4000 nucleotide pairs in a correspondingrestriction fragment.

The object of the invention is to modify the genome of PRV in such a waythat a virus is obtained which is a-virulent, does not induce latentinfection, is genetically stable and, therefore, does not increase invirulence after animal passage, and possesses good immunogenicproperties.

In order to achieve this object the genome was fragmented by means ofrestriction enzymes, and the fragments were separated by using agarosegel electrophoresis and glycerol velocity gradients. The individualfractions were then tested for biological activity by means oftransfection in pig kidney cells. Transfection with intact DNA gave riseto virus production from 5-10 ng DNA per culture bottle. Transfectionwith combined restriction fragments in some cases yielded virus at about50 ng per culture bottle. The viruses produced by the transfected cellswere characterized. It appeared that on transfection, withoutpreliminary ligation, small deletions occurred around the restrictionsites of the enzyme with which the genome had been fragmented. Asexpected, a deletion is tolerated only at a limited number of sites.

By means of the above-described analyses two areas of the PRV genometolerating deletions have been defined. Viruses the genome of whichshows one or more of these deletions appear to be substantially lessvirulent than the parent virus. The modified viruses show clearlyaltered in vitro growth properties. The infection proceeds more slowlyand the cytopathologic effect is clearly different from that of theparent strains. Also in vivo, the deletion mutants appear to behavequite differently. After intranasal inoculation piglets showed only aslight temperature increase, without any further clinical phenomena.During the infection the virus could be reisolated from the saliva.

It was further found that the presence of a functional thymidine kinasegene may lead to undesirable latency of the virus. Consequently, thedeletion mutants according to the invention may possess a deletion inthe thymidine kinase gene.

The invention relates to a live deletion mutant of a herpesvirus, thegenome of which differs from the genome of the parental strain whichconsists of about 160,000 nucleotide pairs and is characterized by arestriction map typical for Pseudorabies virus DNA, for example by therestriction map of FIG. 1, in which the rectangles represent invertedrepeats and the vertical lines represent the cleavage sites of therestriction endonucleases HindIII, KpnI, BamHI and BgIII, which genomecomprises a small unique sequence U_(s) having a restriction map typicalfor the small unique sequence of Pseudorabies virus DNA, for example therestriction map depicted in FIG. 2, in which the arrows represent thecleavage sites of the restriction endonucleases indicated in FIG. 2, bythe presence of one or more of the following deletions:

(a) A deletion in the inverted repeats situated between the cleavagesites of KpnI (between fragment E, or fragment H, respectively, andfragment K+M), and the cleavage sites of BamHI (between BamHI fragments5 and 8);

(b) A deletion in the region which is hatched in the restriction map ofthe small unique sequence (FIG. 2);

(c) A deletion in the thymidine kinase gene.

Deletion (a) preferably comprises about 100 nucleotides, and deletion(c) preferably comprises about 100-500 nucleotides.

In the above-mentioned definition of the deletion mutants according tothe invention, the genomes of the mutants are defined as genomesdiffering from the parental strain genomes by the presence of one, twoor three deletions. In view of the known variability of the parentalPseudorabies virus strains the invention is not limited to mutantsderived from parental strains having genomes corresponding exactly withthe restriction map of FIG. 1. Also, the restriction maps of the smallunique sequence U_(s) of the parental strains may differ from therestriction map of FIG. 2, although the genomes of all of the parentalstrains contain a similar unique sequence. However, all parental strainsare Pseudorabies virus strains inducing the clinical symptoms ofPseudorabies.

The invention also relates to vaccines containing a live virus asdefined above. The vaccines are prepared .[.ina.]. .Iadd.in a.Iaddend.usual way.

Deletion mutants having the above-described properties are obtained byintroducing deletions into the viral genome, by means of molecularbiological techniques, namely in one of the following ways:

(a) Digesting cloned or uncloned subgenome fragments with restrictionendonucleases and then religating part of the so generated fragmentsthereby deleting a fragment or fragments in the respective subgenomefragment, introducing a fragment so modified, optionally after ligationwith the rest of the overlapping or not-overlapping genetic informationof the viral genome, into susceptible cells, so as to produce themodified virus.

(b) Digesting cloned or uncloned subgenome fragments with restrictionendonucleases and effecting a deletion at the cleavage site by treatingthe fragments with an exonuclease and, optionally after religation withthe rest of the overlapping or not-overlapping genetic information ofthe viral genome, introducing the modified fragments into susceptiblecells, so as to produce the modified virus.

(c) Digesting cloned or uncloned subgenome fragments with restrictionendonucleases and effecting a deletion at the cleavage site bytransfecting the generated fragments (without preliminary ligation),together with the rest of the overlapping or not-overlapping geneticinformation of the viral genome, into susceptible cells. These cellswill produce the modified virus. The deletion will be effectedspontaneously during the transfection procedure.

The following example illustrates the construction of the deletionmutants according to the invention, as well as their biologicalproperties. The new mutant strains 2.4N3A and 2.8N3A have been depositedat the Phabagen Collection of the Rijksuniversiteit at Utrecht, VakgroepMoleculaire Celbiologie, Transitorium 3, Padnalaan 8, 3584 CH Utrecht,Netherlands. Deposit numbers PC PV 2 and PC PV3, respectively. Further,the strains have been deposited at the Collection Nationale De CulturesDe Micro-Organismes (C.N.C.M.), Institut Pasteur, Paris. In saidcollection, the Deposit Number of Strain 2.4N3A is I 351, and theDeposit Number of Strain 2.8N3A is I 352.

EXAMPLE Construction of mutants

The construction of the various deletion mutants of pseudorabies virus(PRV) strain NIA-3 is outlined in FIG. 3. NIA-3 virion DNA was digestedto completion with the restriction endonucleases BamHI and HindIII. Theproducts of the digestions were fractionated on preparative agarose gelsand fragments were isolated by electro-elution. The HindIII B and BamHI7 fragments were inserted into the plasmid vector pBR322 and clonedaccording to established procedures resulting in plasmids pN3HB andpN3B7, respectively. Plasmid pN3HB was made by ligating the HindIIIfragment, and plasmid pN3B7 was made by ligating the BamHI 7 fragmentwith pBR322 using T4 ligase. The plasmid pN3B7 (11.1 kbp) carrying theBamHI fragment was used to introduce deletions into the NIA-3 genome. Toaccomplish this, pN3B7 was linearized by partial BalI digestion (45 min37° C., 0.3 units BalI per μg DNA). Full-length DNA molecules wereisolated by agarose gel electrophoresis and electro-elution. Thesemolecules were digested with the exonuclease Bal31. The optimumconditions for digestion (0.18 kbp DNA/min) were 57 nM DNA and 20 unitsper ml of Bal31 exonuclease at 37° C. Aliquots were taken at 7, 12.5 and18.5 min after addition of the exonuclease, and the digestion productswere separated according to size on a low melting agarose gel. DNAfragments of the desired length (8.3 to 9.0 kbp) were selected. DNAmolecules were treated with T4 polymerase, religated with T4 DNA ligaseand introduced into Escherichia coli strain HB101 by standard methods.Of the Ampresistant/Tetsensitive colonies plasmid DNA was isolated. Thesize and position of the deletions were determined by digestion with therestriction endonucleases MluI, BglII, SphI and PvuI. Three recombinantplasmids with different deletions (2.1, 2.4 and 2.8 kbp) were selectedfor further manipulation. They had retained the MluI site at mapposition 0.886 and the BglII site at position 0.856, but had lost theMluI site at position 0.876 (FIG. 3). The MluI-BglII fragments(interrupted lines) of the pN3B7 derivatives with deletions of 2.1, 2.4and 2.8 kbp were isolated and ligated to the 30 kbp MluI-BglII fragmentof plasmid pN3HB. The resulting plasmids pHB 2.1, pHB 2.4 and pHB 2.8were characterized by restriction endonuclease analysis.

For transfection, the HindIII A fragment of NIA-3 virion DNA and theviral inserts of the plasmids pHB 2.1, pHB 2.4 and pHB 2.8, cleaved withHindIII, were purified by two consecutive 5-40% glycerol gradients (20mM Tris-hydrochloride pH 8.0, 1M NaCl, 10 mM EDTA). The purity of theHindIII A fragment and of the viral inserts was verified by restrictionendonuclease cleavage and Southern blot analysis. Subsequently, afourfold molar excess of each of the viral plasmid inserts was mixedwith the HindIII A fragment of NIA-3 virion DNA. The DNA mixtures (500ng) were transfected into secondary porcine kidney cells (PK-2) by thecalcium phosphate coprecipitation technique (Virology 52 (1973),456-467).

The reconstituted viruses obtained from positive transfections werepurified by three rounds of plaque picking and their genomes werecharacterized by Southern blot analysis.

Biological characterization of deletion mutants

The parental strain NIA-3, the avirulent strain NIA-4, and the deletionmutants 2.4N3A and 2.8N3A were examined with respect to their biologicalproperties in mice and pigs.

The mean time to death (MTD) in mice upon inoculation with strains ofPRV was used as a parameter to group strains according to virulence(Archives of Virology 63 (1980), 107-114). In general, virulent PRVkills mice faster than attenuated strains of PRV, although exceptionshave been noted. As can be seen from Table 1, the MTDs of the differentstrains varied considerably. The deletion mutants 2.4N3A and 2.8N3Aexhibited significantly (Wilcoxon rank test, p<0.05) longer MTDs thanthe NIA-3 strain, indicating that a marker for virulence in mice ispresent in the U_(S) and/or repeat region. Also the difference in MTDbetween mutants 2.4N3A and 2.8N3A is significant (p<0.05) suggestingthat the virulence for mice is influenced by the position and/or size ofthe deletion in the U_(S) region.

Pigs from the Dutch landrace "minimal-disease" herd of the CentralVeterinary Institute, which are free from Pseudorabies, were used todetermine the virulence of the deletion mutants in the natural host. Asshown in Table 1, the deletion mutants derived from the NIA-3 strain hada markedly reduced virulence for pigs. The parental NIA-3 strain killed2 to 5 pigs. In the surviving animals intense depression, anorexia andfever (>40° C.), beginning on post inoculation day (PID) 2 was observed.From PID 5 to 7 most pigs showed nasal discharge and dyspnoea.Furthermore, they developed mild to severe neurological signs.Reconstituted NIA-3 without a deletion in the U_(s) region, but with asmall deletion in the repeat .[.unti.]. .Iadd.unit .Iaddend.at theposition of the HindIII site, behaved similar to NIA-3, indicating thatthe deletion in the repeat is not of importance for the virulence inpigs. In contrast, pigs inoculated with mutant 2.4N3A were only slightlydepressed and had a somewhat reduced appetite between PID 2 and 5. Themean body temperatures remained below 40° C. The only sign of disease inpigs inoculated with mutant 2.8N3A was a rise in body temperature: meantemperatures .Iadd.of .Iaddend.40.3, 40.4 and 40.1 on PID 3, 4, and 5,respectively. These results clearly demonstrate that the presence of thedeletions in the U_(S) region of PRV is associated with a reducedvirulence in both mice and pigs. Pigs inoculated with NIA-4 strain alsoshowed a somewhat reduced appetite on PID 4. The virulence of thisstrain for pigs did not change when its HindIII B fragment was replacedby the deleted HindIII B fragment (Δ2.4) of the virulent NIA-3 strain.This shows that the deletion present in the U_(S) region of NIA-4 is themajor factor determining the reduced virulence of the NIA-4 strain. Thegroup's mean growth arrest period is considered to be an importantparameter for the severity of illness. Table 1 shows that in pigsinoculated with NIA-3 virus a mean growth arrest period of 9 days wasobserved, whereas in pigs inoculated with any of the other mutants nogrowth arrest was noticed.

                  TABLE 1                                                         ______________________________________                                        Biological characteristics of deletion mutants of PRV                                   Pigs                                                                                                    Virus   Anti-                                   Mouse             Growth Fe-  excretion                                                                             body                              Virus MTD       Death   arrest ver  pos  mean titre                           ______________________________________                                        NIA-3 50 ± 4 2       9      4    90   3.64 2.85                            NIA-4 170 ± 29.0                                                                           0       0      0    ND   ND   1.13                            2.4   73 ± 7.0                                                                             0       0      0    80   2.65 1.65                            N3A                                                                           2.8   60 ± 8.5                                                                             0       0      3    50   2.20 1.62                            N3A                                                                           ______________________________________                                    

To determine the virulence in mice, groups of eight, 6 to 7-week-oldBALB/c mice were subcutaneously inoculated with 10⁶ plaque forming units(PFU) of PRV. The mice were monitored at intervals of 6 h for 10 days.The mean time to death (MTD) values are given with standard deviation.

To determine the virulence in pigs, groups of five, 10-week-old pigswere inoculated intranasally with a dose of 10⁵ PFU of PRV, byinstilling 0.5 ml into each nostril. The growth arrest period wasdefined as the number of days needed to regain the group's mean weightof post inoculation day (PID) 3. Pigs that died were not included inthis calculation.

Days fever: the average number of days in which body temperatures wereabove 40° C.

Virus excretion: Oropharyngeal fluid (OPF) samples were collected dailyfor ten days after vaccination. The sampling procedure and the virusassay was as described in Veterinary Quarterly 4 (1982), 49-56.

Pos: percentage of positive OPF samples.

.[.Means.]. .Iadd.Mean .Iaddend.titre: means virus titres are expressedas log₁₀ PFU/ml of OPF.

Antibody titre: the neutralizing antibody titre on PID 21 was expressedas the log₁₀ of the reciprocal of the final serum dilution thatinhibited cytopathic effect in 50% of the cultures.

The levels of virus excretion after inoculation, which gives anindication of the capacity of the virus to replicate in the upperrespiratory and/or digestive tract, were also monitored. Pigs infectedwith virulent PRV generally shed much larger quantities of virus forlonger periods than pigs inoculated with attenuated PRV. As shown inTable 1 virtually all oropharyngeal fluid (OPF) samples of pigs givenNIA-3 strain contained virus. In addition, the .[.means.]. .Iadd.mean.Iaddend.virus titre in OPF-samples was the highest in these animals.The virus excretion of pigs given mutants 2.4N3A or 2.8N3A wassubstantially reduced. The pigs surviving the infection with thevirulent NIA-3 strain .[.shows.]. .Iadd.showed .Iaddend.more than 10fold higher .[.means.]. .Iadd.mean .Iaddend.neutralizing antibody titresat 3 weeks post inoculation than the NIA-3 derived deletion mutants. TheNIA-4 strain induced the lowest antibody titre. However, pigs inoculatedwith the deletion mutants were well protected against a challenge withvirulent PRV: Intranasal vaccination with the deletion mutants preventedthe occurrence of clinical signs upon challenge with the virulent NIA-3strain (Table 2). No growth arrest was observed and the mean bodytemperature of pigs inoculated with 2.4N3A and 2.8N3A remained below 40°C. Although protected against clinical illness, the pigs shed virulentvirus via the OPF. However, in comparison with control pigs thepercentage of virus containing samples collected for 10 days afterchallenge was markedly reduced. In addition, the mean titres of PRV inthe OPF were 3 to 5 logs lower than in control pigs (Table 2).

                  TABLE 2                                                         ______________________________________                                        Evaluation of protection induced by deletion mutants                                                               Virus                                           Antibody         Growth       excretion                                Virus  titre    Death   arrest Fever pos   mean                               ______________________________________                                        2.4 N3A                                                                              2.25     0       0      0     20    0.51                               2.8 N3A                                                                              2.04     0       0      0     52    2.00                               controls                                                                             --       0       20     8     100   5.27                               ______________________________________                                    

A group of five sero-negative pigs and two groups of pigs vaccinatedwith the PRV deletion mutants 2.4N3A and 2.8N3A (see Table 1) wereintranasally challenged with 10⁵ PFU of virulent NIA-3 virus at the ageof 16 weeks. Antibody titres were monitored at the day of challenge, 6weeks after vaccination.

We claim:
 1. A live deletion mutant of a pseudorabies virus .[.derivedfrom a.]. .Iadd.obtained by introducing, by means of recombinant DNAtechniques, one or more deletions into the genome of a virulent.Iaddend.parental strain having a parental genome of about 160,000nucleotide pairs, said parental genome comprising(a) two invertedrepeats, each of which contains cleavage sites for the restrictionendonucleases BamHI and KpnI, BamHI digestion of the parental genomeyielding two pairs of restriction fragments derived entirely from thesequence of the inverted repeats, the larger fragment of each pair beingdesignated as BamHI fragments 5; and KpnI digestion of the genomeyielding two fragments, designated KpnI fragment E and .[.kpnI.]..Iadd.KpnI .Iaddend.fragment H, each of said KpnI fragments containingthe distal end of one of the inverted repeats; (b) a unique sequencelocated between the inverted repeats, said unique sequence comprising aBamHI restriction fragment of about 7 kilobase pairs in lengthdesignated BamHI fragment 7, said BamHI fragment 7 containing a cleavagesite for the restriction endonuclease SalI, wherein cleavage at the SalIsite produces two unequal fragments from BamHI fragment 7, one fragmentof about five kilobase pairs in length and the other of about twokilobase pairs in length; and (c) a gene for thymidine kinase, saiddeletion mutant .[.being characterized by one or more deletions, saiddeletions being located in(i) a portion of an inverted repeat which iscommon to BamHI fragment 5 and either KpnI fragment E or KpnI fragmentH; (ii).]. .Iadd.having a deletion in .Iaddend.the about five kilobasepair SalI fragment of BamHI fragment 7, .[.or (iii) both.]. .Iadd.suchthat the mutant is less virulent that the parental strain.Iaddend...[.2. Virus according to claim 1, characterized in that deletion (i)comprises about 100 nucleotides..].
 3. Live Pseudorabies virus deletionmutants according to claim 1, which are derived from NIA-3 strain ofPRV.
 4. Deletion mutant 2.4N3A according to claim 3 as deposited at thePhabagen Collection at Utrecht, Netherlands, deposit number PC PV2. .Deletion mutant 2.8N3A according to claim 3 as deposited at the PhabagenCollection at Utrecht, Netherlands, deposit number PC PV3.
 6. Vaccinecontaining a live virus mutant according to one of claims 1, .[.2.]..Iadd.or .Iaddend.3-5.
 7. A live deletion mutant according to claim 1,including a further deletion in the thymidine kinase gene.
 8. A livedeletion mutant according to claim 7, wherein the deletion in thethymidine kinase gene is from 100 to 500 nucleotides in length. .Iadd.9.Virus according to claim 1, having a further deletion in a portion of aninverted repeat which is common to BamHI fragment 5 and either KpnIfragment E or KpnI fragment H. .Iaddend. .Iadd.10. Virus according toclaim 9, including a further deletion in the thymidine kinase gene..Iaddend.